There is considerable controversy about the amount of normal surrounding breast tissue that should be excised in Breast Conservation Therapy (BCT) 8-13, resulting substantial surgeon-specific variation in re-excision rates (0%-70%)8. Ideally, surgical margins would be defined using objective biological criteria as opposed to an arbitrary physical distance. To achieve this personalized margin, it is necessary to understand all factors contributing to local recurrence. While local recurrences are thought to be caused by residual cancer cells outside the surgical margin14-16, our data suggest that cells in histologicaly normal tissue up to 1 cm away from breast tumors may also have the potential to contribute to local recurrence. The epithelial cells demonstrate evidence of Hallmarks of Cancer alterations, and the fibroblasts share characteristics with Cancer Associated Fibroblasts (CAFs), which are known to promote tumorigenesis in adjacent cells. Since National Guidelines suggest a 2 mm surgical margin lumpectomy 12, and field cancerized tissue can extend 1 cm from the margin, it often remains in a woman after surgery. Our previous studies have identified TGF-- related properties as the key differences between Tumor Adjacent Histologically Normal tissue up to 1 cm from the tumor margin (TAHN-1) and truly normal breast tissue18. For example, the epithelial cells in TAHN-1 tissues exhibit markers and properties of epithelial to mesenchymal transition (EMT), a key endpoint of TGF- signaling 18. Additionally, the stroma exhibits TGF--driven properties such as a-SMA-positive myofibroblasts and a dense extracellular matrix (ECM) enriched in fibrillar collagen and MMP218. Interestingly, we have shown that TAHN-1 fibroblasts, when grown in primary culture, secrete exosomes that induce EMT in epithelial cells, indicating a potential mechanism for the TGF--related properties observed in TAHN-1 tissues. Based on these observations, we hypothesize that tumor adjacent tissues contain fibroblasts that secrete exosomes which induce EMT through TGF- signaling in epithelia. Aims 1 and 2 will involve interfering with individual molecules in the TGF- pathway in TAHN-1 epithelial cells and in exosome-treated MCF10a cells, respectively. We will do this by utilizing an siRNA multiplex array (96 well). We will then verify these key molecules in 3D culture as well as in vivo. We will silence these key molecules using shRNA constructs in TAHN-1 epithelial and exosome-treated MCF10a cells. We will then grow them in 3D Matrigel culture and measure for disrupted apicobasal polarity. To verify these molecules in vivo, we will use shRNA to silence these key molecules in TAHN-1 epithelial and exosome-treated MCF10a cells and inject into the humanized mammary fat pad together with different populations of fibroblasts. Aim 3 will involve identification of TGF--related molecules differentially expressed in TAHN-1 epithelia, fibroblasts, exosomes, and exosome-treated epithelia. We will perform RNAseq on the fibroblast and epithelial populations, as well as MCF10a cells treated with exosomes. We will also analyze the contents of the exosomes using miRNA analysis and proteomics.